Molecular mimicry between Zika virus and central nervous system inflammatory demyelinating disorders: the role of NS5 Zika virus epitope and PLP autoantigens

Background  Evidence indicates a strong link between Zika virus (ZikV) and neurological complications. Acute myelitis, optic neuritis, polyneuropathy, and encephalomyelitis that mimic inflammatory idiopathic demyelination disorders (IIDD) after ZikV infection have been reported in Brazil. Objective  The present study aims to investigate the possible occurrence of molecular mimicry between ZikV antigens and Multiple Sclerosis (MS) autoantigens, the most frequent IIDD of the central nervous system (CNS). Methods  A retrospective cohort study with 305 patients admitted due to suspected arbovirus infection in Rio de Janeiro was performed, all subjects were submitted to neurological examination, and a biological sample was collected for serologic and molecular diagnostic. Bioinformatics tools were used to analyze the peptides shared between ZikV antigens and MS autoantigens. Results  Of 305 patients, twenty-six were positive for ZikV and 4 presented IDD patterns found in MS cases. Sequence homology comparisons by bioinformatics approach between NS5 ZikV and PLP MS protein revealed a homology of 5/6 consecutive amino acids (CSSVPV/CSAVPV) with 83% identity, deducing a molecular mimicry. Analysis of the 3D structures revealed a similar conformation with alpha helix presentation. Conclusions  Molecular mimicry between NS5 Zika virus antigen and PLP MS autoantigens emerge as a possible mechanism for IDD spectrum in genetically susceptible individuals.


INTRODUCTION
Over 80% of Zika Virus (ZikV) infections in humans are asymptomatic. Typical symptoms can include rash, fever, joint pain, and conjunctivitis for a period of 7 days. The outbreak of ZikV has increased the occurrence of long term neurological complications, such as Guillain-Barré syndrome, acute flaccid paralysis, and meningoencephalitis. 1 In addition, ZikV was detected by serology in cerebrospinal fluid (CSF), molecular and histopathological analysis of the brain, and amniotic fluid of microcephalic fetuses. 2,3 ZikV has also been associated with central nervous system (CNS) inflammatory demyelinating disorders (IDD) including optic neuritis, 4 neuromyelitis optica spectrum disorders (NMOSD), 5 transverse myelitis and acute disseminated encephalomyelitis (ADEM). 2 Our group has recently published a case in which the coexistence of the virus in the CNS of an MS patient led to the development of an ADEM-like episode. 6 Besides its direct neurotropic effect, 7 it is believed that ZikV may function as a trigger leading to the development of an immune-mediated injury against many parts of the CNS. 8 ZikV has already been related to the development of several autoimmune conditions. 9 In Guillain-Barre syndrome (GBS), for example, the molecular mimicry between glycolipids and surface molecules of the virus has explained the majority of cases. 10 Interestingly, ZikV is commonly associated with magnetic resonance imaging (MRI) lesions distributed in neurological examination, and a biological sample was collected for serologic and molecular diagnostic. Bioinformatics tools were used to analyze the peptides shared between ZikV antigens and MS autoantigens. Results Of 305 patients, twenty-six were positive for ZikV and 4 presented IDD patterns found in MS cases. Sequence homology comparisons by bioinformatics approach between NS5 ZikV and PLP MS protein revealed a homology of 5/6 consecutive amino acids (CSSVPV/CSAVPV) with 83% identity, deducing a molecular mimicry. Analysis of the 3D structures revealed a similar conformation with alpha helix presentation. Conclusions Molecular mimicry between NS5 Zika virus antigen and PLP MS autoantigens emerge as a possible mechanism for IDD spectrum in genetically susceptible individuals.
Obejtivo O presente estudo tem como objetivo investigar a possível ocorrência de mimetismo molecular entre antígenos do ZikV e autoantígenos da Esclerose Múltipla (EM), a DDII mais frequente do sistema nervoso central (SNC). Métodos Foi realizado um estudo de coorte retrospectivo com 305 pacientes internados por suspeita de infecção por arbovírus no Rio de Janeiro, todos os indivíduos foram submetidos a exame neurológico e coleta de amostra biológica para diagnóstico sorológico e molecular. Ferramentas de bioinformática foram usadas para analisar os peptídeos compartilhados entre antígenos do ZikV e autoantígenos da EM. Resultados Dos 305 pacientes, vinte e seis foram positivos para ZikV e 4 apresentaram padrão IDD encontrado em casos de EM. As comparações de homologia de sequência por abordagem de bioinformática entre a proteína NS5 ZikV e PLP EM revelaram uma homologia de 5/6 aminoácidos consecutivos (CSSVPV/CSAVPV) com 83% de identidade, deduzindo um mimetismo molecular. A análise das estruturas 3D revelou uma conformação semelhante com apresentação em alfa-hélice. space and time, regarding heterogeneous gadolinium enhancement, as seen in the MS criteria. 11 Moreover, serum positivity for autoantibodies against myelin oligodendrocyte glycoprotein (MOG), a specific antibody against the myelin sheath was recently associated with ZikV. 12 As many radiological and clinical aspects of ZikV infection may mimic IIDD, patients can be misdiagnosed. MS is the most frequent IIDD of the CNS, 13 and several evidences have shown that molecular mimicry is a possible epigenetic mechanism in genetically susceptible individuals. 14 To investigate the mechanisms of ZikV induced neurological manifestations, it is essential to use various reproducible in vitro models and bioinformatics tools capable of recapitulating complex neurodevelopmental disorders, in an attempt to find specific targets. The molecular mechanisms underlying these conditions in adults are not clear. Focusing on the MS-like pattern, the present study investigated the possible occurrence of molecular mimicry between ZikV antigens and MS autoantigens. The underlying rationale is that shared peptides between pathogen and human host may lead to a break in immune tolerance through a cross-reactivity phenomenon. 15

Study population and biological samples
A retrospective cohort study was performed with patients admitted in neurology service of three university hospitals and referred by Laboratório Central Noel Nutels (LACEN) in Rio de Janeiro. This work was approved by the National Council for Ethics in Research (CAAE 69411317.6.0000. 5258). All subjects signed an informed consent agreeing to participate in this research. From 2016 to 2019, 305 patients with suspected arbovirus infection were evaluated by a multidisciplinary team. Complete physical and neurological examination was performed and, when necessary, MRI was requested. Biological sample (blood, urine, and CSF) was collected on admission and, according to clinical indication, tested by serology and/or ZikV molecular diagnostic.

Sequence analysis
Peptide sharing between ZikV antigens and MS autoantigens was analyzed as follows: A viral polyprotein library was constructed using the major viral antigens reported in the literature and protein sequences available in NCBI Protein Reference Sequences (https://www.ncbi.nlm.nih. gov/protein). An MS autoantigen library was constructed at random through UniProtKB Database (www.uniprot.org/) using 'Multiple Sclerosis' as a keyword. The result was filtered and only the proteins confirmed as autoantigens were collected. ZikV polyproteins and MS autoantigens identified are outlined in ►Table 1. The two libraries were analyzed for matches using BLASTP (https://blast.ncbi. nlm.nih.gov/Blast.cgi) and sequence alignment was done using EMBOSS (https://www.ebi.ac.uk/Tools/psa/emboss_ water/).

Antigenic prediction
To confirm whether the NS5 ZikV sequence studied has antigenic properties, VaxiJen version 2.0 (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) was used. A threshold antigenic score of 0.5 was defined in order to filter probable non-antigenic sequences. Vaxijen server performs alignment-independent prediction, which is based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties.

3D comparative modelling
The 3D models were built using the Swiss-Model, an online modeling server (https://swissmodel.expasy.org/). The template modeling scores (TM-scores) and root mean square deviations (RMSDs) of the NS5 ZikV and PLP MS threedimensional overlap were calculated using TM-Align.

Inflammatory demyelinating disorder phenotypes in patients with ZikV infection
A total of 305 patients were evaluated. 26 were positive for ZikV and the remaining were diagnosed with either Dengue or Chikungunya. Out of the ZikV positive patients, 4 were classified as having IDD of the CNS requiring differential diagnosis with MS. Clinical examination, imaging, electrophysiologic, and laboratory findings of these patients are exposed in ►Table 2 and ►Figures 1-4.
Patient 1 presented with headache, optical neuritis, and cervical myelitis associated with a cervical lesion (►Figure 1 E) and asymptomatic multifocal brain lesions on MRI, one of which had gadolinium enhancement. This distribution of brain lesions, paired with positive oligoclonal bands (OCB) found on CSF analysis, resembles the pattern usually found in MS (►Figure 1 A-D and ►Table 1).
Patient 2 had a diagnosis of acute flaccid paraplegia 11 days after a viral prodrome, and 3 months later developed tetraparesis associated with longitudinal extensive transverse myelitis (►Figure 2C and 2D), centrally located (►Figure 2 C3, D3), with focal tapering of the cervical/dorsal transition on sagittal STIR (►Figure 2 C1), resembling the extension and sequelae areas usually seen in NMOSD. Furthermore, the lesion had anterior horn involvement (►Figure 2 D4).
Patient 3 presented with tetra paresis and ataxia associated with brain lesions mainly affecting the brainstem on axial T2 images, including the posterior aspect of the mesencephalon (►Figure 3E), pons (►Figure 3F) and the medial cerebellar peduncle (►Figure 3G).
Patient 4 presented with optic neuritis and multifocal myelitis with cervical and dorsal lesions, as usually found in a first manifestation of MS (►Figures 4A and 4B).

Sequence sharing between ZikV polyproteins and MS autoantigens
The bioinformatics approach identified an 83% identity between the NS5 antigen of ZikV and PLP MS autoantigen, deducing the molecular mimicry among them. Although statistically non-significant, it was also possible to observe a 67% identity between NS3 antigen of ZikV and MOG MS autoantigen. The identity results between all sequences are depicted in ►Table 3. In addition, sequence analysis of NS5 using VaxiJen version 2.0 resulted in a score of 0.5091, confirming the antigenicity of the sequence studied.

Structural conformation between NS5 ZikV and PLP MS
In order to predict the 3D structures conformation of the two proteins, TM-Align was used to align them. As Blast P showed us a high identity between a particular region of PLP and NS5, a structural conformation was performed only with that region where the corresponding high identity was obtained PLP 131-198 and NS5 281-325 (►Figure 5B). The CSAVPV sequence which is 83% identity by BlastP, obtained a TM-score of 0.47071 and RMDS of 2.39 and is in the alpha helix structure of both proteins.

DISCUSSION
Several studies have shown that IDD in CNS can be triggered by viral infection or immunizations. After a variable period of incubation, myelin destruction undergoes courses of remission and exacerbation. MS is a most common disease that compromises CNS myelin sheath. 16 Viral infection can trigger autoimmune diseases through different mechanisms: molecular mimicry, epitope spreading, bystander activation, superantigen production, and inadequate activation of an immune response. 17 Molecular mimicry can be defined as similar structures shared by a host epitope and microorganism or environmental proteins. 17 Using bioinformatics tools, common sequences and structural homology between Chikungunya virus (ChikV) E1 glycoprotein and human HLA-B27 molecule were identified. In addition, the peptides derived from ChikV glycoprotein E1    induced significant inflammation in C57BL/6J mice. 18 Based on proteomic studies and sequence analysis, some evidence has also shown that Dengue Hemorrhagic Fever may be caused by molecular mimicry between different coagulation molecules with prM, E, and NS1 viral proteins. 19 Furthermore, it is already widely proposed that cross-recognition of common viral peptides with myelin antigens induces a molecular mimicry involved in MS development, especially in genetically susceptible individuals. 14 Zika Virus has 7 non structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). Each of them has a paper on the antagonism to the innate immunity. However, the NS5 protein stands out here, as it is the most conserved protein in the flavivirus proteome and it can modulate the host immune response during ZikV infection. 20 NS5 is an antagonist of the interferon response in the host human system and promotes the degradation of STAT2 in ZikV. This same mechanism is well reported in DenV. The importance of NS5 in host immune response modulation and viral replication makes it an attractive target for developing broadly acting antiviral inhibitors. 21 Zika fever is a self-limited disease, still, less than 5% of symptomatic patients may develop neurological manifestations. 22,23 Although patient 1 developed neurological manifestations five days after the first symptoms of viral infection, it was only possible to make the ZikV diagnosis after sixty days, thus explaining the IgM negativity in serum.
A recent case report identified concurrent GBS and ADEM in a 24-year-old woman who developed acute ZikV infection. The authors postulate this case was para-infectious, induced   by neurotropism and activation of an immune response against ZikV. 24 This same mechanism is probably involved in the development of this NMOSD phenotype in our patient 2. Patient 3 could be classified as having a clinical isolated syndrome (CIS) with a high risk of conversion to MS due to the distribution and number of T2 white matter lesions. Although the optical neuritis pattern resembles the one of NMOSD, the spinal cord lesions are MS-like. Lucchese et al. 2016, observed that ZikV antigens are commonly involved in microcephaly and GBS. 129 immunopositive epitopes are reported as having peptide overlap with human proteins that may relate to demyelination and axonal neuropathies. This indicates that cross-reactivity with human proteins might contribute to the mechanisms linking ZikV infection to GBS. 10 The IDD phenotype attrib-uted to ZikV infection seems to mimic MS manifestations. Molecular mimicry is assessed in this study by investigating homologous regions between ZikV antigens and human MS autoantigens using bioinformatics tools. Sequence homology comparisons between NS5 ZikV and PLP MS protein revealed a homology of 5/6 consecutive amino acids CSSVPV/CSAVPV (►Figure 5A). A study that performed antigenic B-cell epitopes prediction found an antigenic peptide from position 528 to 539 (NAICSSVPVDWV) of ZikV NS5, which had the maximum residual score of 1.203 and might present a preliminary set of peptides for future vaccine development against ZikV. 25 Calculating the TM-score of NS5 ZikV and PLP MS 3D structures demonstrated that both proteins are in almost the same fold, both are in alpha helix and they have topological similarity (►Figure 5B). 26 Interestingly, ZikV African (MR766) lineage strain, revealed exactly the same Human PLP sequence (CSAVPV), and recombinant NS5 proteins from Africa and from Brazil revealed similar levels of RNA synthesis. 27 It is already known that the MR766 strain is more virulent and causes more severe brain damage than the current Asian lineage and dengue virus. 28 When inoculated subcutaneously in adult transgenic mice (knockout) C57BL/6 Stat2 -/-MR766 strain induces short episodes of severe neurological symptoms, followed by lethality. Furthermore, this strain was able to induce higher levels of inflammatory cytokines and markers associated with cellular infiltration into the brain of infected mice. 29 Li et al. 2019, observed that MR766 strain and epidemic Brazilian (BR15 and ICD) ZikV strains are different in viral attachment to host neuronal cells, viral permissiveness, and replication, as well as in the induction of cytopathic effects. 30 Autoreactivity to PLP in patients with MS has been investigated in human and animal model by various groups worldwide. 31 A recent study involving PLP's Epitopes involved in MS, found CSAVPV (in PLP 161-177 residues) among the most immunogenic regions of PLP. 32 In addition, the crystal structure of the NS5 ZikV protein reveals a conserved domain conformation of Flaviviruses, a genus that includes a variety of human pathogens such as dengue virus, yellow fever virus, WNV, Spondweni virus, and the Japanese encephalitis virus. 33 So, the presence of high identity between NS5 ZikV and PLP, an autoantigen widely implicated in the pathogenesis of MS, 34 leads us to postulate that molecular mimicry may have a role in the development of inflammatory demyelinating damage, a hallmark of the IDD produced by this genus of virus.
Both genetic and environmental factors have been shown to contribute to the pathogenesis of autoimmune diseases. It is well-established that the HLA-DR15 haplotype bears the strongest association with MS. 35 In a Brazilian study, it was observed that the presence of HLA-DRB1 Ã 1501 allele confers an ethnicity-dependent MS susceptibility in Caucasian patients and that the HLA-DQB1 Ã 0602 allele confers an ethnicity independent susceptibility. 36 Using HLA class II transgenic (Tg) mice, several studies have demonstrated HLA-DR-dependent disease following immunization by MBP, PLP, or MOG. 37,38 However, it was observed that HLA-DRB1 Ã 1501 Tg mice were refractory to disease induction by overlapping PLP peptides, while HLA-DQB1 Ã 0602 Tg mice were susceptible to disease induction by PLP 139-151 and PLP [175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193][194] peptides. 39 It has been seen that Both PLP 139-151 and PLP [178][179][180][181][182][183][184][185][186][187][188][189][190][191] epitopes are key targets of T-cells, and are increased in MS patients versus healthy controls. 40 However, this does not mean that PLP 161-177 residues are not encephalitogenic-related, but that they need further animal and human model studies. Therefore, PLP autoimmunity and HLA haplotype have been strongly associated with lesion localization, as well as remission and relapse rates in MS. 41 In conclusion, the concept of molecular mimicry remains a viable hypothesis for understanding the genetics, epigenetics, and environmental involvement in the pathogenic mechanisms of IDD. Studies using bioinformatics tools further encourage the identification of molecules that could be used in the development of either diagnostic or prognostic biomarkers. We found that NS5 ZikV presented a high identity with PLP MS autoantigen, and both are structurally similar to alpha helix chains. These findings may justify IDD CNS manifestations following ZikV infection, as in the 4 cases here reported. Further investigation is required to understand whether PLP 161-177 residues are encephalitogenic and how the recognition of NS5 epitopes by HLA molecules drives the pathogenic T-cell autoimmune response in vivo.